Mice models of human prostate cancer progression

ABSTRACT

The present invention provides an immune deficient mouse having a human prostate xenograft of locally advanced or metastatic prostate cancer and uses thereof.

[0001] This application is a continuation-in-part of U.S. Ser. No.08/732,676, filed Oct. 15, 1997, the contents of which is herebyincorporated by reference in its entirety.

[0002] Throughout this application, various publications are referencedwithin parentheses. Full citations of these publications may be found atthe end of the specification immediately preceding the claims. Thedisclosures of these publications are hereby incorporated by referenceherein in their entireties.

BACKGROUND OF THE INVENTION

[0003] Prostate cancer is the most common cause of cancer in men. In1996, 317,000 new cases of prostate adenocarcinoma were diagnosed andover 41,400 men died of the disease (Karp et al., 1996). Only lungcancer has a higher mortality. The chance of a man developing invasiveprostate cancer during his lifetime is 1 in 6 or 13.4%. At the age of50, a man has a 42% chance of developing prostate cancer and 2.9% ofdying from the disease. While advances in early diagnosis and treatmentof locally confined tumors have been achieved, prostate cancer isincurable once it has metastasized. Patients with metastatic prostatecancer on hormonal therapy will eventually develop anandrogen-refractory (androgen independent) state that will lead todisease progression and death.

[0004] The major cause of morbidity and mortality from prostate canceris the result of androgen-independent metastatic tumor growth. As aresult, there is great interest in defining the molecular basis foradvanced staged disease with the hope that these insights may improvethe therapeutic options for these patients. However, progress in thisarea has been difficult for a number of reasons. For example, theavailability of prostate tissue for molecular studies is limited becausemost prostate tumors are small Moreover, there is tremendousheterogeneity within surgical prostatectomy tumor samples, it isdifficult to reducibly culture prostate cancer explants in vitro, andthere are a limited number of immortalized prostate cancer cell lines.

[0005] There is, therefore, an interest in finding alternativeprocedures which will allow for stable growth of prostate cancer tissue,which in turn would allow for the investigation of the progression ofprostate cancer in vivo, provide a stable supply of prostate cancertissue and provide a model for metastatic expansion of prostate cancerwhich accurately simulates or mimics the biology of the disease.

[0006] There is also a need for more reliable and informative stagingand prognostic methods in the management of advanced prostate cancer.Clinically staging prostate tumors relies on rectal examination todetermine whether the tumor remains within the borders of the prostaticcapsule (locally confined) or extends beyond it (locally advanced), incombination with serum PSA determinations and transrectal ultrasoundguided biopsies. However, none of these techniques has proven reliablefor predicting progression of the disease.

[0007] The primary sites of prostate cancer metastasis are the regionallymph nodes and bone. Bone metastases occur in sites ofhematopoietically active red bone marrow, including lumbar vertebralcolumn, ribs, pelvis, proximal long bones, sternum and skull. Bonymetastases of prostate cancer differ from those of other tumors thatcommonly colonize in bone in that they are characterized by a net gainin bone formation (osteoblastic) rather than resorption predominant inbone metastases of breast cancer and melanoma.

[0008] Until recently, bone metastasis was thought to be a late stage indisease progression However, the recent development of highly sensitivetechniques (such as RT-PCR for prostate specific genes) to detectprostate cancer cells has revised this notion. Prostate cancer cellshave been detected in the peripheral blood and bone marrow of patientswith advanced stage disease using RT-PCR assays for PSA mRNA (Ghosseinet al., 1995; Seiden et al., 1994; Wood et al., 1994; Katz et al., 1994)or immunomagnetic bead selection for PSA protein (Brandt et al., 1996).When positive, these tests show that prostate cancer cells representabout 0.1-1.0% of the circulating blood cells Moreover, it is now clearthat small numbers of prostate cancer cells circulate in the peripheralblood and lodge in the bone marrow even in patients with early stage.low risk disease (Olsson et al., 1997; Deguchi et al., 1997; Katz etal., 1996). Interestingly these cells tend to disappear in most patientsfollowing radical prostatectomy (Melchior et al., 1997). These resultssuggest that the primary tumor site is a constant source for seeding themarrow, and that only a small subset of these cells have the capacity togrow into a metastatic lesion. This concept is consistent with estimatesfrom animal models for other tumor types that only about 1 in 10,000circulating cancer cells are able to lodge in and productively colonizeother organs (Fidler et al., 1990).

[0009] The factors involved in advanced prostate cancer progression tobone metastasis are poorly defined. Anatomic, local bone/marrow andtumor cell factors are all believed to play a role. Baston described theextensive vertebral venous system that consists of a network oflongitudinal, valveless veins that run parallel to the vertebral columnand form extensive, direct anastomoses with-the veins of the ribs,pelvis and brain (Baston. 1942). Prostate cancer cells enteringprostatic veins may be transported via this plexus directly to theseorgans without entering the inferior vena cava of passing through thelungs. This hypothesized mechanism of metastasis both by clinicaldocumentation of patterns of prostate cancer metastasis compared toother tumors and by animal models wherein occlusion of inferior venacava during tail vein injection of tumor cells increased the incidenceof vertebral metastasis (Nishijima et al., 1992; Coman and DeLong,1951).

[0010] Although the vascular anatomy is an essential component of thespread of prostate cancer to bone, it cannot fully explain the selectivepattern of all skeletal metastases Bone, which receives 5-10% of thecardiac output, is a more frequent metastatic site than would beexpected from blood-flow criteria (Berettoni and Carter, 1986). Bonemarrow consists of two clearly identifiable components: the hematopoeticcells which comprise the majority of the cellular elements, and stromalcomponent that is formed of highly vascular connective tissue. Thehematopoetic cells are transient in the bone marrow; upon maturationthey move into the blood stream. The stroma, however. remains and servesas a scaffolding upon which the hematopoetic cells can differentiate andmature. One of the important factors in prostate cancer cells arrestingin these sites is likely their adhesion to the bone marrow stroma. Ithas beer demonstrated both in vitro and in vivo that tumor cells willpreferentially adhere to the stromal cells of the organs to which theymetastasize (Haq et al., 1992; Netland and Zetter, 1985; Zetter et al.,1992). When rat prostate cancer (MatLyLu) cells were injected into theleft ventricle of syngenic rats, vertebral body metastases developed.these metastases were then collected, disaggregated and reinjected Celllines established after 6 similar passages through animals adheredstrongly and preferentially to bone marrow stroma and endothelial cells(Haq et al, 1992). A similar approach has increased the incidence ofmetastasis from the LNCaP prostate cancer cell line in immune deficientmice (Thalmann et al., 1994).

[0011] It is critical that appropriate in vivo models for prostatecancer bone metastasis be developed to more fully explore themechanistic aspects of this process. To date, most work in this area hasfocused on three human prostate cancer cell lines PC-3, DU-145, andLNCaP (Lee et al., 1993). All three grow a subcutaneous nodules inimmune deficient mice, and sublines with variable metastatic propertieshave been derived (Shervin et al., 1988, 1989; Wang and Sterarns, 1991;Kozlowski et al., 1988). However, none of these sublines has been shownto reproducibly give rise to osteoblastic lesions typical of prostatecancer. A major limitation of the DU-145 and PC-3 cell lines is the lackof prostate specific antigen (PSA) and androgen receptor (AR) expression(Kaighn et al., 1979; Gleave et al., 1992), which raises regardingrelevance to clinical prostate cancer. The LNCaP cell line is androgenresponsive and expresses PSA, but contains a mutation in the androgenreceptor which alters ligand specificity.

SUMMARY OF THE INVENTION

[0012] The invention provides animal xenograft models of human prostatecancer progression capable of simulating or mimicking the development ofprimary tumors, micrometastasis, and the formation of osteoblasticlesions characteristic of late stage disease. The model may be used tostudy the stagewise progression of prostate cancer. In this regard, theinvention replicates the process of cell migration from the primarytumor site to distant sites of micrometastasis, including bone marrow,as well as the development of macrometastatic osteoblastic bone lesionsfrom micrometastatic precursors. The models are also capable ofduplicating the clinical transition from androgen dependent to androgenindependent tumor growth characteristic of advanced prostate cancerpatients undergoing androgen ablation therapy. In addition. methods forpropagating prostate cancer cells within the various stages of prostatecancer as well as methods for isolating and expanding stage-specificprostate cancer cell populations are also provided. Further, theinvention provides a unique. serially-passaged, androgen-sensitiveprostate cancer cell line which expresses PSA, wild-type androgenreceptor, and prostate acid phosphatase. The models and methods of theinvention provide a system for studying the molecular biology ofprostate cancer, evaluating the influence that various genes andtherapeutic compounds have on distinct stages of disease progression,assessing the metastatic potential of prostate cancer cells, anddesigning patient-specific therapeutic regimens.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1. Molecular analysis of prostate cancer xenografts for humanDNA content and expression of prostate specific antigen (PSA). FIG. 1A:DNA-PCR analysis of genomic DNA isolated from xenografts using primersspecific for the human β-globin gene. Each sample shown was obtainedfrom late passage xenografts. The LAPC-5 sample was obtained at passage4 when the human tumor was overgrown by a tumor of murine origin. Thetwo LAPC-4 samples were obtained from androgen dependent (“ad”) andandrogen independent (“ai”) sublines FIG. 1B: RT-PCR analysis of totalRNA using primers specific for human PSA and human or murine β-actin (“%human cells” refers to the percent of LNCaP cells which are diluted into10⁵ mouse cells). A dilution series of human prostate cancer LNCaP cellsinto murine NIH 3T3 cells is shown on the left side of the figure, withthe percentage of LNCaP cells varying from 100% to 0.0%. The resultsfrom the three LAPC xenografts are shown on the right.

[0014]FIG. 2. Photographs of immunohistochemical analysis of the LAPC-4xenograft. showing expression of PSA. Paraffin sections offormalin-fixed tissue from the original tumor sample obtained at thetime of surgery (top row) and the LAPC-4 xenograft (bottom row) werestained with hematoxylin and eosin (left), a control antibody (middle)and an antibody specific for human PSA (right).

[0015]FIG. 3. Bar graphs showing androgen sensitivity of the LAPC-3 andLAPC-4 xenografts in vivo. Equal size implants of the LAPC-3 and LAPC-4xenografts were passaged simultaneously into male or female mice andexamined weekly for the formation of tumors.

[0016]FIG. 4. Regression and regrowth of LAPC-4 tumors followingcastration. FIG. 4A: Line graph showing typical results from two animalsin the cohort whose tumor sizes were equivalent at 4 weeks. The timecourse for tumor development in a female mouse is shown for comparison.FIG. 4B: Bar graph showing the average tumor size (+/−standard error)from the entire cohort of intact and castrated male mice. The data fromeach animal are expressed as tumor size relative to the 4 week timepoint.

[0017]FIG. 5. Line graph showing limiting dilution analysis of LAPC-4engraftment in male mice.

[0018]FIG. 6. Photographs showing detection of micrometstatic disease inmice bearing LAPC-4 xenografts. Total RNA was isolated from the murinetissues indicated and analyzed for the expression of PSA (a) or β-actin(b) using RT-PCR. The results from the tumor and various tissues ofthree representative mice are shown. Tissues from a fourth mouse(control SCID) were analyzed as a negative control. The signal can bequantified by comparison to with LNCaP (lane 1). No RNA was added to thenegative control sample (lane 2).

[0019]FIG. 7. Photographs showing detection of LAPC-4 cells in bone byimmunohistochemistry after 2 weeks. Frozen sections of the tibia of miceinjected with LAPC-4 cells were stained with an antibody tocytokeratin-18 (bottom panel) or an isotype control antibody (toppanel). The four cells staining red are LAPC-4 cells.

[0020]FIG. 8. Photographs showing LAPC-4 causes bone lesions.Hematoxylin and eosin sections of the tibia are shown at 4, 6 and 8weeks following intratibial injection of LAPC-4 cells. Panel A shows asmall focus of tumor formation adjacent to normal bone andhematopoiesis. Panels B and C show progressive increase in new boneformation in response to surrounding tumor cells.

[0021]FIG. 9. Radiographic evidence of osteoblastic bone lesions inducedby LAPC-4. X-rays of mice were performed at 8 weeks post injection ofLAPC-4 cells in the tibia (right panel). The bone shows evidence oferosion of the cortex with enhanced bone density in the marrow cavitydue to osteoblastic activation.

[0022]FIG. 10. Flow cytometry analysis of LAPC-4 cells stained withanti-galectin-6 antibody. showing the expression level relative to anisotype control antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Immune Deficient Animal Hosts

[0024] Severe combined immune deficient (SCID) mice are the preferredanimal host utilized in the practice of the invention. Various otherimmune deficient mice, rodents or animals may be used, including thosewhich are deficient as a result of a genetic defect. which may benaturally occurring or induced, such as, for example, nude mice, Rag 1and/or Rag 2 mice, and the like, and mice which have been cross-bredwith these mice and have an immunocompromised background. The deficiencymay be, for example. as a result of a genetic defect in recombination, agenetically defective thymus or a defective T-cell receptor region.Induced immune deficiency may be as a result of administration of animmunosuppressant, e.g. cyclosporin, removal of the thymus, etc Varioustransgenic immune deficient mice are currently available or can bedeveloped in accordance with conventional techniques. Ideally, theimmune deficient mouse will have a defect which inhibits maturation oflymphocytes, particularly lacking the ability to rearrange the T-cellreceptor region. Female, male, castrated or uncastrated mice may beemployed, depending upon whether one is interested in studying theeffect of the availability of androgens on the course of the tumorgrowth. In the particular and preferred embodiments described herein,C.B. 17 scid/scid mice are used. In addition to mice, immune deficientrats or similar rodents may also be employed in the practice of theinvention.

[0025] Models That Simulate Advanced Prostate Cancer

[0026] One aspect of the invention provides murine xenograft modelswhich simulate or mimic human prostate cancer from primary tumorformation. Also provided are methods for propagating advanced stagehuman prostate tumor tissue as subcutaneous xenografts in immunedeficient mice. In the practice of the invention, prostate cancerxenografts may be established in immune deficient mice by thesubcutaneous implantation of fresh human prostate cancer explantssurgically removed from patients with locally advanced or metastaticprostate cancer. The site of implantation may be into any subcutaneoussite which will permit blood supply to reach the implant, such as theflanks of the host animal. Tissue from primary prostate tumors as wellas from sites of lymph node, lung. bone, and other organ metastases maybe used to establish the prostate cancer xenografts of the invention.Prostate tumor explants may be introduced in conjunction with a basementmembrane composition, such as Matrigel (U.S. Pat. No. 5,508,188), anextracellular matrix preparation which has been shown to enhance thegrowth of epithelial tumors in vivo (including prostate cancer cells)(Lim et al., 1993; Noel et al., 1992; Pretlow et al., 1991), as well asother similar types of compositions. Once established, the xenografttumors grow to considerable size, providing substantial tissue volumesfor further use. Xenografts of the invention retain the human phenotypeas determined by human β-globin expression, express human prostatespecific antigen (PSA), and retain androgen sensitivity and metastaticgrowth characteristics reflective of the clinical situation.

[0027] As used herein, the term “locally advanced prostate cancer” and“locally advanced disease” mean prostate cancers which have extendedthrough the prostate capsule. and are meant to include stage C diseaseunder the American Urological Association (AUA) system, stage C1-C2disease under the Whitmore-Jewett system, and stage T3-T4 and N+ diseaseunder the TNM (tumor, node, metastasis) system. In general, surgery isnot recommended for patients with locally advanced disease, and thesepatients have substantially less favorable outcomes compared to patientshaving clinically localized (organ-confined) prostate cancer. Locallyadvanced disease is clinically identified by palpable evidence ofinduration beyond the lateral border of the prostate.,or asymmetry orinduration above the prostate base. Locally advanced prostate cancer isdiagnosed pathologically following radical prostatectomy if the tumorinvades or penetrates the prostatic capsule, extends into the surgicalmargin, or invades the seminal vesicles

[0028] As used herein, the terms “metastatic prostate cancer” and“metastatic disease” mean prostate cancers which have spread to regionallymph nodes or to distant sites, and are meant to include stage Ddisease under the AUA system and stage T×N×M+ under the TNM system. Asis the case with locally advanced prostate cancer, surgery is generallynot indicated for patients with metastatic disease, and hormonal(androgen ablation) therapy is the preferred treatment modality.Patients with metastatic prostate cancer eventually develop anandrogen-refractory state within 12 to 18 months of treatmentinitiation, and approximately half of these patients die within 6 monthsthereafter. The most common site for prostate cancer metastasis is bone.Prostate cancer bone metastases are, on balance, characteristicallyosteoblastic rather than osteolytic (i e., resulting in net boneformation). Bone metastases are found most frequently in the spine,followed by the femur, pelvis, rib cage, skull and humerus. Other commonsites for metastasis include lymph nodes, lung, liver and brain.Metastatic prostate cancer is typically diagnosed by open orlaparoscopic pelvic lymphadenectomy, whole body radionuclide scans,skeletal radiography, and/or bone lesion biopsy.

[0029] This and other aspects of the invention described herein providetools for studying the pathogenesis and treatment of advanced prostatecancer. For example, immune deficient mice bearing subcutaneous (andother) xenografts may be used to evaluate the effect of various prostatecancer treatments (e.g., therapeutic compositions, gene therapies,immunotherapies, etc.) on the growth of tumors and progression ofdisease Xenograft cells may be used to identify novel genes and geneswhich are differentially expressed in prostate cancer cells, or toanalyze the effect such genes have on the progression of prostatecancer. For example, the genetic compositions of prostate cancer cellsfrom xenografts having differing androgen sensitivities (e.g., androgendependent vs. androgen independent) may be compared to each other aswell as to the genetic compositions of normal prostate cells. Likewise,the genetic compositions of micrometastatic prostate cancer cells may becompared to those of metastatic prostate cancer cells. Various nucleicacid subtraction and sampling techniques may be used for this purpose,including, for example, representational difference analysis (RDA) Inaddition, prostate cancer xenograft cells may be used for theintroduction of various genetic capabilities, including the introductionof various genes, antisense sequences, ribozymes, regulatory sequenceswhich enhance or repress the expression of endogenous genes, and soforth.

[0030] In addition, this aspect of the invention provides methods forpurifying prostate cancer cells from the heterogeneous mixture of cellstypical of human prostate cancer biopsy material, further providingmethods for generating greater quantities of tumor cells for subsequentuse and analysis. In one embodiment, the method for purifying prostatecancer cells comprises implanting human prostate cancer biopsy materialsubcutaneously into a SCID or other immune deficient mouse and allowingthe implanted material to grow as a xenograft in the mouse. The purifiedhuman prostate cancer cells are obtained by harvesting the xenograft.Xenografts may be expanded and further purified by serial propagation inadditional immune deficient mice or by propagation in short term cellculture. Single cell suspensions of xenograft tumor tissue or culturedcells may be used to orthotopically seed intraprostatic tumors, bonetumors or other organ tumors. Xenograft tumor tissue and cellpreparations may be frozen and viably recovered for later use.

[0031] The invention also provides subcutaneous prostate cancerxenografts welch retain stable prostate cancer cell phenotypes throughmultiple passages in SCID mice. Various embodiments are provided,including androgen dependent and androgen independent xenografts,xenografts which express prostate specific antigen (PSA) at clinicallyreflective levels, xenografts which express wild-type androgen receptor(AR). and xenografts which exhibit chromosomal abnormalities. Stillother embodiments include xenografts which retain all of the foregoingcharacteristics as well as xenografts that model the progression toandrogen independent disease. These and other embodiments of theinvention are described in more detail by way of the examples whichfollow. As described in Example 1, a number of subcutaneous xenograftswere successfully established from tumor tissue explants taken from theprostate gland and from bone, lymph, and lung metastases of patientswith stage C or D prostate cancer These xenografts grow and passage inSCID mice with high frequency and retain definitive characteristics ofhuman prostate cancer, even in late passages. One xenograft, designatedLAPC-4, has been adapted to tissue culture as a stable cell line and hasbeen in continuous culture for 18 months.

[0032] Xenografts such as the LAPC-4 xenograft described in Example 1are of particular interest. Similar to prostate tumors isolated directlyfrom patients, LAPC-4 cells retain expression of prostate specificantigen (PSA), androgen receptor (AR), and prostatic acid phosphatasethrough more than 20 passages. Moreover, the LAPC-4 xenograft is uniqueamong prostate cancer model systems since its AR contains no mutationsin the DNA or ligand binding domains and AR expression is retained inandrogen independent LAPC-4 sublines. In addition, the LAPC-4 xenograftmodels the transition from androgen-dependent to androgen-independentdisease as well as the development of micrometastatic disease. Forexample, LAPC-4 tumors passaged in male mice retain androgen-dependentgrowth characteristics, whereas tumors passaged in castrated males orfemale mice acquire a stable androgen-independent phenotype. Thesesublines can be easily expanded using the methods of the invention toprovide ample tissue for molecular and biochemical analysis of eventsassociated with androgen-independent growth. There are few otherexperimental models for androgen-dependent prostate cancer growth.Published reports include the widely used LNCaP cell line (Lim et al.,1993; Gleave et al., 1992) and two recently described xenografts, CWR22(Weinstein et al., 1994) and LuCaP23 (Lin et al., 1996). The LAPC-4xenograft is unique because tumors placed under the selective pressureof androgen deprivation reproducibly evolve to an androgen-independentstate, providing an opportunity to evaluate the molecular changesassociated with androgen-independence over time and directly test theirfunctional importance.

[0033] This aspect of the invention also provides assays for determiningthe function or effect of various genes on prostate cancer cells. In oneembodiment, the assay comprises isolating prostate cancer cells from aprostate cancer xenograft (e.g., subcutaneous. intraprostatic),transducing the cells with the gene of interest such that the transducedcells express or overexpress the gene, establishing a subcutaneous orintraprostatic xenograft tumor in a SCID or other immune deficient mousewith the transduced cells and evaluating the growth of the resultingxenograft. The effect of expressing the gene on the growth of thexenograft may be determined by reference to a control xenograftestablished with untransduced prostate cancer cells, preferably isolatedfrom the same parental xenograft. In another embodiment, the assaycomprises generating a prostate cancer xenograft (e.g., subcutaneous,intraprostatic), transducing the cells of the xenograft with the gene ofinterest in vivo, and evaluating the growth of the xenograft. whereinthe effect of the gene on the growth of the xenograft may be determinedby reference to a control xenograft.

[0034] Similarly, the invention provides assays for determining theeffect of candidate therapeutic compositions or treatments on the growthof prostate cancer cells In one embodiment, the assay comprises applyingthe composition or treatment to a SCID or other immune deficient mousebearing a subcutaneous human prostate cancer xenograft and determiningthe effect of the treatment on the growth of the xenograft In anotherembodiment, a SCID or other immune deficient mouse bearing anintraprostatic xenograft is used to determine the effect of thecomposition or treatment.

[0035] This aspect of the invention may also have various clinicalapplications, including using the model in a method to assess prognosisof a patient with locally advanced or metastatic prostate cancer. Forexample, in one embodiment, the method comprises implanting a prostatetumor sample from the patient into an immune deficient mousesubcutaneously, and allowing the implanted sample to grow as a xenograftin the mouse. The rates of xenograft growth may be used as a prognosticindicator. The results of such analysis may assist a treating oncologistin determining how aggressively to treat a patient.

[0036] Models That Simulate Prostate Cancer Micrometastasis

[0037] Another aspect of the invention provides models and methods forsimulating and studying the process of micrometastasis in human prostatecancer. SCID mice bearing subcutaneous prostate cancer xenografts showevidence of circulating prostate cancer cells. Thus, this modelduplicates the process of cell migration from the primary tumor to thebone marrow and other distant sites of micrometastasis. As detailed inExample 3, 100% of male mice inoculated subcutaneously with xenograftLAPC-4 cells developed localized subcutaneous tumors within 4 to 6 weekswithout evidence of bony metastasis. However, when these animals wereexamined for the presence of micrometastatic disease, up to 50% of themice had detectable prostate cancer cells in bone marrow and blood.Using the same semi-quantitative RT-PCR assay that has been applied tolarge surveys of prostate cancer patients, micrometastatic prostatecancer cells were found at levels comparable to about 0.1 to 1.0 % ofthe total mouse bone marrow. Similar results were obtained byimmunohistochemical analysis for PSA expression. Thus, subcutaneousgrowth of prostate cancer xenografts mimics the clinical observationthat prostate cancer cells circulate in the blood and lodge in the bonemarrow, even in early stage disease.

[0038] In one embodiment, simulating or mimicking prostate cancermicrometastasis comprises establishing a subcutaneous prostate cancerxenograft in a SCID or other immune deficient mouse and allowing thetumor to grow for a time sufficient to permit the detection of prostatecancer cells in the peripheral blood of the mouse. The presence ofmicrometastasis is monitored by detecting prostate tumor cells whichhave migrated to the lympahtic and/or vascular system, bone, lung,liver, and/or other sites distant from the primary xenograft site.Detection of such cells may be accomplished by, for example, assayingfor the presence of human PSA mRNA in the peripheral blood using anRT-PCR assay for PSA mRNA (such as the assay described in Example 3)

[0039] In another embodiment, simulating prostate cancer micrometastasiscomprises preparing a single cell suspension of prostate cancer cellsfrom a subcutaneous xenograft tumor grown in a SCID (or other immunedeficient) mouse, followed by intraprostatic (orthotopic) injection ofthe single cell suspension into another SCID (or other immune deficient)mouse. The intraprostatic tumor is allowed to grow for a time sufficientto permit the detection of prostate cancer cells in the peripheral bloodof the mouse or in other sites distant from the orthotopic tumor. Singlecell suspensions prepared from cultured xenograft cells may also be usedfor intraprostatic (orthotopic) implantation.

[0040] This aspect of the invention also provides a framework fortesting the effect of certain variables on the development ofmicrometastasis. Such variables may include the presence or absence ofhormones or other growth-modulating factors in the environment of thetumor, the expression status of various genes within the tumor cells,etc. For example, the rate of micrometastatic of androgen dependent andandrogen independent xenograft variants may be evaluated. Such anevaluation is described in Example 3, using the androgen dependent andindependent sublines of the LAPC-4 xenograft, demonstrating asignificantly higher rate of micrometastasis in mice bearing theandrogen independent LAPC-4 xenografts.

[0041] In this regard, the invention provides assays for determining thefunction or effect of various genes on the progression of prostatecancer micrometastasis. In one embodiment, the assay comprises isolatingprostate cancer cells from a prostate cancer xenograft (e.g.,subcutaneous, intraprostatic), transducing the cells with the gene ofinterest such that the transduced cells express or over-express thegene. using the transduced cells to establish a subcutaneous orintraprostatic xenograft tumor in a SCID or other immune deficientmouse, and evaluating the presence and level of micrometastatic diseaseby detecting prostate cancer cells in blood, lymph nodes, and/or othersites distant from the site of the primary xenograft tumor The effect ofexpressing the gene on the rate of micrometastasis may be determined byreference to a control xenograft established with untransduced prostatecancer cells preferably isolated from the same parental xenograft. Inanother embodiment, the assay comprises generating a prostate cancerxenograft (e.g., subcutaneous. intraprostatic), transducing the cells ofthe xenograft with the gene of interest in vivo, and evaluating thepresence and levels of micrometastatic disease, wherein the effect ofexpressing the gene on the rate of micrometastasis may be determined byreference to a control xenograft.

[0042] Similarly, the invention provides assays for determining theeffect of candidate therapeutic compositions or treatments on theprogression to micrometastatic disease. In one embodiment, the assaycomprises applying the composition or treatment to a SCID mouse bearinga subcutaneous human prostate cancer xenograft, and determining theeffect of the treatment on micrometastasis by monitoring the presenceand levels of prostate cancer cells in the peripheral blood, lymphnodes, bone marrow, and/or other sites distant from the xenograft. Inanother embodiment, a SCID or other immune deficient mouse bearing anintraprostatic xenograft is used to determine the effect of thetreatment on micrometastasis.

[0043] This aspect of the invention may also have various clinicalapplications, including using the model in a method to assess prognosisof a patient with locally advanced or metastatic prostate cancer. Forexample, in one embodiment, the method comprises implanting a prostatetumor sample from the patient into an immunocompromised mousesubcutaneously and allowing the implanted sample to grow as a xenograftin the mouse. The rates of xenograft growth and the development ofmicrometastasis may be used as prognostic indicators. The results ofsuch analysis may assist a treating oncologist in determining howaggressively to treat a patient.

[0044] Models That Simulate Metastatic Prostate Cancer

[0045] Another aspect of the invention provides models and methods formimicking and studying the development of macrometastatic osteoblasticbone lesions (bone metastasis) in prostate cancer. Subcutaneous growthof xenograft tumors results in detectable micrometastasis, indicatingthat cells from xenograft tumors in SCID mice have the ability to exitthe site of primary tumor growth, circulate in blood, and lodge in thebone marrow, reflecting the human clinical situation.

[0046] In one embodiment, simulating the development of prostate cancerbone metastasis comprises injecting a single cell suspension of prostatecancer cells prepared from a subcutaneous prostate cancer xenograftgrowing in SCID (or other immune deficient) mouse into the prostate ofanother SCID (or other immune deficient) mouse host. and allowing theresulting orthotopic tumor to grow for a time sufficient to permit thedetection of bone metastasis in the mouse. Alternatively, subcutaneousprostate cancer xenografts may be established with such single cellpreparations and allowed to grow. Detection of bone metastasis may beaccomplished by various means including histologically,immunohistochemically, and radiographically.

[0047] Subcutaneous and orthotopic tumors typically grow quickly,reaching a size which demands that the host animal be sacrificed withinabout 4-6 weeks. Therefore alternative methods which increase the numberof prostate cancer cells in the bone marrow, thereby obviating thislimitation, are also provided. In one embodiment, a single cellsuspension prepared from xenograft tumor cells, or from xenograft cellsin tissue culture, is injected directly into the bone marrow cavity(e.g., tibial) of a SCID or other immune deficient mouse. Thedevelopment of micrometastasis, bone tumor growth, and osteoblasticactivity may be monitored in various ways, including byimmuno-histochemistry and in situ hybridization of bone sections or byradiographic imaging.

[0048] As described in Example 6, a single cell suspension of 10,000xenograft tumor cells prepared from a subcutaneous tumor was injectedinto the tibia of a SCID mouse A small subset of the injected cells wasdetectable at 2 weeks, followed by small foci of bone tumor growth in afew isolated areas at 4 weeks, followed by extensive macroscopic bonetumor growth, destruction of bone cortex, and net new bone formation by6-8 weeks. Accordingly, cells isolated from the human prostate cancerxenografts of the invention are capable of proliferation in themicroenvironment of the SCID mouse bone marrow cavity.

[0049] The foregoing method provides an excellent model for simulatingthe formation of osteoblastic bone lesions and the progression to thisstage of the disease. The model may be used not only to study themolecular and cellular events involved in the progression of this stageof prostate cancer, but also to test the effect of various candidatetherapeutic genes, proteins and other compounds. In addition, the modelmay be used as an assay for assessing the metastatic and osteoblasticpotential of prostate cancer cells obtained from human patients.

[0050] Accordingly, the invention also provides assays for determiningthe function or effect. of various genes on the progression of prostatecancer bone metastasis. In one embodiment, the assay comprises isolatingprostate cancer cells from a prostate cancer xenograft (e.g.,subcutaneous, intraprostatic, bone), transducing the cells with the geneof interest such that the transduced cells express or over-express thegene. introducing the transduced cells into the bone marrow cavity of aSCID or other immune deficient mouse, and monitoring the bone marrow forthe presence and levels of osteoblastic macrometastatic lesions. Theeffect of expressing the gene on the development and growth of bonemetastasis may be determined by reference to a control animal receivinguntransduced prostate cancer cells, preferably isolated from the sameparental xenograft. In another embodiment, the assay comprisesgenerating a bone marrow xenograft in a SCID (or other immune deficient)mouse by injecting a single cell suspension of prostate cancer cellsprepared from a subcutaneous or intraprostatic xenograft established inanother SCID (or other immune deficient )mouse, transducing the cells ofthe bone marrow xenograft with the gene of interest in vivo, andevaluating the effect of the gene on the presence and levels ofosteoblastic macrometastatic lesions.

[0051] Further, the invention provides assays for determining the effectof candidate therapeutic compositions or treatments on the progressionof prostate cancer bone metastasis. In one embodiment, the assaycomprises applying the composition or treatment to a SCID or otherimmune deficient mouse receiving an intratibial injection of prostatecancer xenograft cells and determining the effect of the treatment onthe progression of bone metastasis by monitoring the tibial bone marrowfor the presence and levels of prostate cancer cells and/or osteoblasticmacrometastatic lesions. The presence of prostate cancer cells in bonemarrow may be detected by various means, including histologically,immunochemically, or by assaying for the presence of PSA mRNA orprotein. The presence of osteoblastic macrometastatic lesions may bedetected using histologic, radiographic, or other imaging techniques.

[0052] This aspect of the invention may also have various clinicalapplications, including using the model in a method to assess theprognosis of a patient with locally advanced prostate cancer, and inparticular, to predict the likelihood that a patient will progress tometastatic disease. For example, in one embodiment, the method comprisesinjecting a single cell suspension prepared from a patient's prostatebiopsy material directly into the bone marrow of an immune deficientmouse and then monitoring the bone marrow for the development of bonelesions. The rate of bone lesion growth and osteoblastic activity may beused as prognostic indicators. The results of such analysis may assist atreating oncologist in determining how aggressively to treat a patientwith locally advanced disease.

[0053] Similarly, the effect of various therapeutic strategies formanaging locally advanced or metastatic disease in a particular patientmay be predicted. For example, the effect of a treatment strategy may bepredicted by applying the treatment to an immune deficient mousereceiving a bone marrow injection of the patient's prostate cancer cellsThe effect of the treatment may be monitored by comparing the rate andextent of bone lesion growth and osteoblastic activity in the test mouseto the corresponding rates in an untreated control mouse receiving acorresponding bone marrow injection. In addition, this method may beused to test the effectiveness of a treatment strategy on androgenindependent prostate cancer cells by using a female or castrated maleimmune deficient mouse in order to select for androgen independentclones in the patient's tumor material. The results of such tests mayassist a treating oncologist in determining which of several alternativetherapies should be used to manage a particular patient's disease.

[0054] Short-Term Culture of Xenograft Tumor Cells

[0055] Xenograft tumor cells may be expanded using short-term in vitrotissue culture techniques well known in the art. In addition, differentclonal populations from a xenograft tumor may be isolated through tissueculture techniques. In this regard, the invention provides methods forpreparing single cell suspensions from xenograft tumor tissue samples.In one embodiment, xenograft tumor tissue is surgically removed from asubcutaneous xenograft tumor, disaggregated, and proteolyticallydigested, using the method described in Example 2 or similar methods.Cells may then suspended in a solution of Matrigel, other basementmembrane compositions, saline, or other buffers. Such preparations areuseful for establishing new tumors in SCID or other immune deficientrecipient mice by, for example, subcutaneous inoculation, intraprostaticinjection, or by injection directly into bone marrow metaphyses. Cellsuspensions may be prepared from subcutaneous, intraprostatic, bone orother orthotopic tumors growing in SCID or other immune deficient mice.

[0056] Methods of Expanding and Purifying Prostate Cancer CellPopulations

[0057] Another aspect of the invention provides methods for expandingadvance prostate cancer cells, methods for preparing relatively purepopulations of prostate cancer cells from heterogeneous populations ofcells, and methods for propagating stage-specific prostate cancer cellsin vivo or in vitro. Primary tumor samples are heterogeneous in theircellular compositions, and are usually contaminated with normal andstromal cells. Moreover, it is difficult to obtain substantialpopulations of prostate cancer cells from human tissue biopsy material.In contrast, cells harvested from subcutaneous prostate tumors growingin SCID mice predominantly comprise prostate cancer cells. Thus, themodels of the invention provide a vehicle for purifying advanced stagehuman prostate cancer cells from heterogeneous biopsy material.

[0058] Serial passage of xenograft tumors in additional mice may be usedto further enhance the prostate cancer specificity of the xenograftcellular composition. Similarly, the ability to serially propagate, suchas by serial propagation, relatively pure human prostate cancer cells inimmune deficient mice provides a means for obtaining large quantities ofdefined prostate cancer cells.

[0059] In one embodiment, tissue harvested from xenograft tumors isenriched for prostate cancer cells by subsequent passage in additionalSCID or other immune deficient mice. In another embodiment, cells fromxenograft tumors may be cultured in vitro. In another embodiment, singlecell suspensions of prostate cancer cells may be prepared from suchcultured cells or directly from xenograft tumor tissue. Single cellsuspensions prepared from protease digested subcutaneous xenografttumors retain the biological properties of the parental tumors. Thesingle cell suspensions may be used to establish, for example, newsubcutaneous tumors, intraprostatic tumors, or bone tumors. As shown bythe experiments set forth in Example 2, as few as 10 xenograft cells canseed a new subcutaneous tumor.

[0060] Selective factors may be added to the environment in which thetumor cell enrichment is being conducted in order to expand cells with aparticular phenotype. For example. the presence of androgen in the invivo environment may be controlled by chemical or surgical castrationmethods well known in the art in order to select for androgen dependentor independent prostate cancer cells. Alternatively, female mice may beused to expand androgen independent cells. Similarly, in an in vitroenvironment, the absence of androgen in growth media may be used toselect androgen independent prostate cancer cells.

[0061] In addition, the presence of cell surface proteins on the tumorcells of subcutaneous xenografts may be used to distinguish and isolatehuman prostate cancer cells from other cells. In particular, antibodiesto cell surface proteins differentially expressed on prostate cancercells (relative to their expression on murine marrow cells) may be usedto isolate prostate cancer cells from xenograft tumor tissue, from cellsin culture. etc. using antibody-based cell sorting or affinitypurification techniques. Most preferred for antibody-based cell sortingare antibodies to cell surface proteins which are human prostate cancerspecific. However, antibodies to other human proteins may be effectivelyemployed provided they do not exhibit significant cross-reactivity withthe murine homolog of the protein. An examples of such a protein ishuman galectin-6.

[0062] The ability to generate large quantities of relatively pureadvanced stage human prostate cancer cells which can be grown in tissueculture or as xenograft tumors in SCID or other immune deficient miceprovides many advantages, including, for example, permitting theevaluation of various transgenes or candidate therapeutic compounds onthe growth or other phenotypic characteristics of a relativelyhomogeneous population of prostate cancer cells. Additionally, thisfeature of the invention also permits the isolation of highly enrichedpreparations of human prostate cancer specific nucleic acids inquantities sufficient for various molecular manipulations. For example,large quantities of such nucleic acid preparations will assist in theidentification of rare genes with biological relevance to prostatecancer disease progression.

[0063] Another valuable application of this aspect of the invention isthe ability to analyze and experiment with relatively pure preparationsof viable prostate tumor cells cloned from individual patients withlocally advanced or metastatic disease. In this way, for example, anindividual patient's prostate cancer cells may be expanded from alimited biopsy sample and then tested for the presence of diagnostic andprognostic genes, proteins, chromosomal aberrations, gene expressionprofiles, or other relevant genotypic and phenotypic characteristics,without the potentially confounding variable of contaminating cells. Inaddition, such cells may be evaluated for neoplastic aggressiveness andmetastatic potential in the subcutaneous, orthotopic, and bone tumormodels of the invention. This aspect of the invention provides a meansfor testing alternative treatment modalities with a view towardscustomizing optimal. patient-specific treatment regimens. Similarly,patient-specific prostate cancer vaccines and cellularimmunotherapeutics may be created from such cell preparations.

[0064] The prostate cancer models of the invention further providemethods for isolating stage-specific prostate cancer cells, includingmicrometastatic and osteoblasLIc prostate cancer cells. In oneembodiment, micrometastatic cells are isolated from hematopoetic tissuessuch as bone marrow or blood using antibody-based cell sorting oraffinity purification techniques. In another embodiment, osteoblasticprostate cancer cells are isolated from the bone marrow of SCID or otherimmune deficient mice bearing osteoblastic bone lesions. The presence ofsuch bone lesions may be detected histologically, immunohistochemically,or radiographically. Stage-specific prostate cancer cells may be furtherexpanded and purified by subsequent replantation into SCID or otherimmune deficient mice. For example, osteoblastic prostate cancer cellsmay be subpassaged in vivo by reinjection into bone marrow or in vitrousing defined bone stroma as a growth substrate.

[0065] As shown by the experimental work presented in Example 3, smallnumbers of micrometastatic prostate cancer cells can be detected in andisolated from the bone marrow of SCID mice bearing subcutaneous prostatecancer xenografts. Although these prostate cancer cells represent lessthan about 1% of the cells in the bone marrow of the host mice, they maybe isolated and expanded using cell purification methods, such as thosediscussed above. In one embodiment, bone marrow harvested from micebearing subcutaneous xenografts is incubated with a human specificmonoclonal antibody to galectin-6 and a secondary antibody conjugated tomagnetic beads. Prostate cancer cells are then isolated using MiltenyiMagnetic Minimacs columns (Sunnyvale, Calif.) to magnetically retainantibody-positive cells in the column while allowing antibody-negativecells to pass to the flow-through. Small numbers of micrometastaticprostate cancer cells isolated in this manner may be expanded in vivo bysubcutaneous inoculation of Matrigel suspended cells into SCID or otherimmune deficient mice. Osteoblastic prostate cancer cells may besimilarly isolated directly from bone marrow lesions.

[0066] The ability to purify and expand stage-specific prostate cancercells may have various clinical applications. For example,stage-specific prostate cancer cells within clinical material may beisolated by using cell sorting or purification techniques and thenexpanded as subcutaneous, intraprostatic, or bone tumors in SCID orother immune deficient mice, depending on the particular objective. Inone embodiment, micrometastasis are isolated from patient serum,formulated into single cell suspensions, and injected intosubcutaneously with the objective of expanding these cells generally. Inan alternative embodiment, the micrometastatic cell preparation isinjected into the bone marrow of a SCID or other immune deficient mousewith the objective of selectively expanding those cells withosteoblastic characteristics Prostate cancer cells passaged in thismanner may become conditioned by various factors within themicroenvironment of the bone marrow and may form osteoblastic lesionswhich may then be harvested for further use or analysis.

[0067] Continuous Cell Lines

[0068] The invention also provides continuous human prostate cancer celllines established by culturing xenograft cell preparations. In oneembodiment, the cell line comprises human prostatic cancer cellscultured from a subcutaneous xenograft. In a specific embodimentdescribed by way of Example 9, the cell line LAPC-4 was established byculturing a single cell suspension prepared from the LAPC-4 xenograft.The LAPC-4 cell line expresses PSA, androgen receptor (AR), and isandrogen dependent. The LAPC-4 cell line has been growing in continuousculture for 1.5 years, and retains phenotypic characteristics whichcorrelate to the human clinical situation more closely than any otheravailable human prostate cancer cell line.

[0069] The cell lines of the invention may be used for a number ofpurposes. By way of example and not by way of limitation, the cell linesmay be used as a source of large quantities nucleic acids and proteins,as a tool for screening and evaluating candidate therapeutic transgenes,proteins and compounds, and as a tool for identifying and isolatingprostate specific or differentially expressed genes. Genes Which mayregulate the growth of prostate cancer can be evaluated byoverexpression in transduced cells grown in vitro or in vivo. Theeffects of genes on micrometastasis and the development of osteoblasticbone lesions may be evaluated in vivo by subcutaneous, intraprostatic,or intratibial inoculation of transduced cells.

EXAMPLES

[0070] The invention is further described and illustrated by way of thefollowing examples and the experimental details therein. This section isset forth as an aid to understanding the invention, but is not intendedto, nor should it be construed as, limiting the invention as claimed.

Example 1 Generation of Subcutaneous Human Prostate Cancer Xenograftsthat Simulate Prostate Cancer Progression

[0071] Materials and Methods

[0072] Patients: All clinical material was obtained from patients withlocally advanced or metastatic (stage C or D) after obtaining informedconsent according to an IRB approved protocol. Most patients hadundergone some form of androgen ablation therapy (medical or surgical)and shown progressive disease at the time the tissue samples wereobtained.

[0073] Animals: C.B.17 scid/scid (SCID) mice were bred at UCLA understerile conditions as previously described (Aldrovandi et al., Nature363:732-736 (1993)). Biopsy material obtained at the time of surgery wasplaced on ice and immediately transferred to the SCID mouse facility forimplantation. A scalpel was used to mince the tissue into 2-3 mm³sections which were then implanted subcutaneously into the flanks ofSCID mice. Mice were anesthetized with methoxyflurane beforeimplantation. Initial implants were performed with 100-200 μl ofMatrigel (Collaborative Research, Bedford, Mass.) injected around theimplant. Matrigel is an extracellular matrix preparation useful forenhancing the growth of epithelial tumors in vivo (Pretlow (1993),supra; Noel et al., Biochemical Pharmacology 43:1263-126⁷ (1992) and Limet al., Prostate 22:109-118 (1993)). Once a xenograft was passaged 2-3times, Matrigel was no longer used for serial propagation Androgenablation was performed by surgical castration under anesthesia. Tumorsizes were determined by weekly caliper measurements of height, widthand depth. Sustained-release testosterone pellets (Innovative Researchof America, Sarasota, Fla.) were implanted subcutaneously, asrecommended by the manufacturer, in some experiments. Xenografts werestored viably in liquid nitrogen by freezing 1-2 mm³ minced tissuesections in DMSO-containing medium.

[0074] PCR Assays, Histology and Immunochemistry: DNA from tumor tissuewas prepared using SDS detergent extraction and proteinase K digestionas described by Sambrook et al., Molecular Cloning. A Laboratory Manual,Cold Spring Harbor Laboratory Press. Edition 2 (1989). RNA was preparedby using a commercially available kit containing guanidine thiocyanateand β-mercaptoethanol (RNAgents Total RNA Isolation System. Promega). Toavoid contamination of gross tissue preparations, the tissue homogenizerand all surgical instruments used at the time of necropsy were cleanedby repeated rinses in HCl, DEPC treated water and ethanol. DNA-PCRassays for human β-globin (Aldrovandi et al., supra and Saiki et al.,Science 230:1350-1354 (1985)) and RT-PCR assays for PSA (Pang et al.,Hum. Gene Ther. 6:1417-1426 (1995)) were performed as previouslydescribed. Briefly, PCR analysis using primers specific for the humanβ-globin gene were performed for 30 cycles with 100 ng of genomic DNAisolated from the LAPC xenografts. One-tenth of each reaction mixturewas analyzed by electrophoresis through agarose gels and visualized bystaining for ethidium bromide Murine 3T3 cells were used as a negativecontrol. The quality of all RNA samples was confirmed by ethidiumbromide staining for ribosomal RNA and by RT-PCR using primers forβ-actin (Pang et al., supra) as a control. Details on the primersequences can be found in the original references. RT-PCR analysis forPSA expression was performed on 100 ng of total RNA using primersspecific for human PSA. The same RNA samples were analyzed using primerswhich recognize human or murine β-actin to confirm equivalent loading ingels. Immunohistochemical staining for PSA was performed usingpolyclonal antisera to PSA (Dako) as described (Hsu et al., Am. J. ClinPath. 75:734-738 (1981)).

[0075] Sequencing Androgen Receptor DNA: Exons 2-8 of the androgenreceptor were sequenced from genomic DNA using intron-specific PCRprimers (Marcelli et al., Mol. Endocrinol. 90:1105-1116 (1990)). PCRproducts were initially screened by SSCP using appropriate positive andnegative controls as described (Sutherland et al., J. Urol. 156. 828-831(1996)). This technique has been shown to detect mutations in prostatecancer clinical specimens even when tumor cells represent only 20% ofthe population used to make genomic DNA. All SSCP abnormalities wereanalyzed by sequencing. Two independent DNA samples were analyzed in twoindependent laboratories to rule out the presence of any mutations.

[0076] Cytogenetics: Tumor tissue was aseptically transported in DMEMgrowth medium supplemented with 10% fetal bovine serum by overnightcourier to the University of Utah for cytogenetic preparation andanalysis. Briefly, tissue was minced and washed in Hanks Balanced SaltSolution (Ca⁺⁺ and Mg⁺⁺ free), resuspended in RPMI medium supplementedwith lot fetal bovine serum, and cells were arrested in metaphase with0.001 μg/ml colcemid for 16 hours. Cytogenetic harvests were carried outusing standard procedures and, following hypotonic (0.075M) KC treatmentand 3.1 methanol/acetic acid fixation, slides were prepared andchromosomes G-banded with trypsin/Wrights stain.

[0077] Results

[0078] Advanced Stage Prostate Cancer Explants Can Be SeriallyPropagated in SCID Mice

[0079] Biopsies of locally advanced or metastatic tumor tissue wereobtained from a total of 15 patients with locally advanced or metastatic(stage C, D1 or D2) prostate cancer who underwent palliative surgicalprocedures due to complications from disease. Biopsy material wasimmediately transferred from the surgical suite to the SCID mousefacility minced into 2-3 mm3 sections and implanted subcutaneously intoSCID mice in the presence of Matrigel. Tumor growth was scored positiveonly if the explant showed 2 sustained a two- to three- fold increase insize. In addition to histologic studies two molecular assays wereperformed on each xenograft to verify the human origin of the tumors.These include a PCR assay on genomic DNA using primers specific for thehuman β-globin gene and a quantitative RT-PCR assay on RNA from tumorsusing primers specific for the human PSA gene. The PSA expression assaywas also used to verify the prostatic origin of the xenografts.

[0080] The results obtained from subcutaneous implantation of tumortissue samples from two separate series of these 15 patients areindividually described below (i.e., the LAPC-1 through LAPC-8 series,and the LAPC-9 through LAPC-15 series).

[0081] LAPC-1 through LAPC-8 Series

[0082] Explants from six of eight patients (named LAPC 1-8 for LosAngeles Prostate Cancer) formed tumors after a latent period whichvaried from 2-10 months (Table 1). The six explants which grew werepassaged into secondary recipients in an attempt to establish permanentxenografts. Two of these (LAPC-1 and LAPC-5) were terminated after 3-4passages because we were unable to detect human DNA or expression of PSAin the tumors. These explants perhaps were overgrown by cells of murineorigin because they contained human DNA content and expressed PSA duringearly passages of LAPC-5 (Table 1, column 6,7).

[0083] The remaining four explants (LAPC-3,4,7 and 8) were successfullypropagated as subcutaneous xenografts in secondary recipients forbetween 4 and 20 (or more) passages. RT-PCR was used to measure thelevels of PSA mRNA expression in comparison with LNCaP, a prostatecancer cell line known to express PSA mRNA and protein. This assay issemi-quantitative and is capable of detecting PSA mRNA expression from100 LNCaP cells diluted into 10⁵ mouse cells (1 in 1000 or 0.1%) (FIG.1B, top panel). Four of the six xenografts (LAPC-3, 4, 5, 8) expressedhuman PSA at levels that varied from 1% to 100% of the level found inLNCaP cells (FIG. 1B, top panel) Simultaneous RT-PCR analysis usingprimers for β-actin confirmed that equivalent levels of RNA were presentin each reaction (FIG. 1B, bottom panel). FIG. 2 shows a histologicalcomparison of the original LAPC-4 tumor sample obtained, at the time ofsurgery to the same tumor after passage as a xenograft in male mice. Thehematoxylin, and eosin stained sections (FIG. 2, left panels) show amonotonous population of anaplastic cells which stain positive for PSAusing immunohistochemical analysis (FIG. 2, right panels). Thesefindings demonstrate that advanced stage prostate cancer explants can beserially propagated in SCID mice and retain definitive tissue specificgene expression. TABLE 1 SUMMARY OF IMPLANTS INTO SCID MICE/LAPC-1THROUGH LAPC-8 SERIES¹ Patient Implant Time interval (disease Biopsy fortumor Human DNA stage) site Growth growth Passages Status² PSA Status³Notes LAPC-1 liver met yes 2 months 5 negative on negative overgrowth by(stage D) passage tumor of murine origin after serial passage LAPC-2lymph no no growth at 2 1 — — — (stage D) node years met LAPC-3 prostateyes 10 months 3 positive positive no PSA (stage D) -channel positivecells TURP outside site of implantation (n = 2) LAPC-4 lymph yes 3months >8 positive positive PSA positive (stage D) node cells in bonemet marrow, spleen, blood in 50% of mice (n = 12) LAPC-5 lymph yes 9months 5 positive, then positive, then overgrowth by (stage D) nodenegative on negative on tumor of met passage 4 passage 4 murine originon passage 4 LAPC-6 prostate no no growth at 9 1 — — — (stage C) monthsLAPC-7 prostate yes 3 months 2 positive negative — (stage C) LAPC-8lymph yes 10 months 2 positive positive no PSA (stage D) node positivecells met outside implantation site (n = 1)

[0084] Two of the xenografts in this series, LAPC-3 and LAPC-4, haveretained constant histologic and molecular features of prostate cancerfor more than 6 and 8 passages. respectively. Both xenografts can befrozen viably as tumor explants and recovered from freezing with nearly100% efficiency. A cell line from the LAPC-4 xenograft was establishedby serial passage-of trypsinized, minced xenograft tissue in Iscove'sgrowth medium supplemented with 20% fetal calf serum. The LAPC-4 cellline has remained established for more than 20 passages and has been incontinuous culture for over 18 months. These cells continue to expressPSA, form tumors in SCID mice, and retain androgen-responsiveness.

[0085] LAPC-9 through LAPC-15 Series

[0086] A second series of xenograft experiments was conducted byimplanting tissue samples from an additional seven prostate cancerpatients with advanced stage (C or D) disease (Table 2). Four of theseseven implants have resulted in the generation of androgen-responsivexenografts which express PSA and which are capable of being propagatedserially in additional mice (LAPC-9, 12, 14, 15). The LAPC-9 xenograft,generated from a bone tumor biopsy of a patient with hormone-refractorymetastatic disease, demonstrates an extremely androgen-sensitivephenotype (PSA levels drop to zero after castration) and has beenpassaged and viably maintained for about 1 year. The LAPC-14 xenograft,generated from a prostate tumor biopsy of a patient with metastaticdisease, demonstrates aggressive growth characteristics and exhibits ahigh degree of androgen-responsiveness (growth is substantially enhancedby the addition of testosterone). TABLE 2 SUMMARY OF IMPLANTS INTO SCIDMICE/LAPC-9 THROUGH LAPC-15 SERIES¹ Patient Time Implant interval forHuman (disease tumor DNA PSA stage) Biopsy site Growth growth PassagesStatus² Status³ Notes LAPC-9 femur yes 5 weeks 5 positive positivepatient hormone- (stage D) tumor refractory androgen dependent LAPC-10prostate no — — — — Gleason 9 (stage C/D) (prostat- ecomy) LAPC-11 femurno — — — — 1 week post lupron (stage D) tumor 2 weeks post flutamideLAPC-12 prostate yes 13 weeks 1 positive positive metastatic (stage D)TURP hormone refractory LAPC-13 trans- no — — — — Gleason 7 (stage C/D)rectal biopsy LAPC-14 prostate yes 4 weeks 2 positive positivemetastatic (stage D) TURP path PSA < 0.2 neg lupron treated androgenresponsive LAPC-15 prostate yes 12 weeks 1 positive positive metastaticto bone (stage D) TURP lupron treated casodex treated X 4 mos

[0087] LAPC-3 and LAPC-4 Xenografts Contain Chromosome Abnormalities

[0088] Extensive cytogenetic studies of human prostate cancer have beendifficult due to heterogeneity of clinical material obtained at surgeryand limited growth of prostate tumor cells in vitro. To determine ifpassage of prostate tumor tissue in SCID mice might facilitatekaryotypic analysis, early passage tumors from the LAPC-3 and LAPC-4xenografts were analyzed using standard cytogenetic techniques. A highmitotic yield was obtained from tumor samples from both xenografts andall metaphase cells contained human chromosomes. Detailed compositekaryotypes are noted in Table 3. The modal chromosome number of LAPC-4was 89, suggesting a hypotetraploid line, whereas the modal chromosomenumber of LAPC-3 was 69, which suggests that this line is near-triploid,yet the presence of four copies of many chromosomes raises thepossibility of reduction from tetraploid. Both xenografts showpreviously reported numerical and structural chromosome abnormalitiessuch as loss of Y and 16 In addition, both xenografts contain a deletionat chromosome 12p12, a karyotypic abnormality that has not beenpreviously reported in prostate cancer. TABLE 3 CYTOGENETIC ANALYSIS OFLAPC-3 AND LAPC-4 XENOGRAFTS Passage number at time of analysis (numberof Number of Modal Xeno- independent metaphases Chromosome graft tumors)analyzed Number Karyotype LAPC-3 passage 2 80 69 68-81, XXY + add (1tumor); (l)(p22), −2, +3, +4 passage 3 +5, del(6)(q21) x2, (2 tumors)+7, +9, +9, −11, del(12)(p12), −13, −13, +14, t(14; 14) (q10; q10), −16,+18, +19, +20 [cp80] LAPC-4 passage 3 44 89 76-92, XX, −Y, (2 tumors)−Y, add(8)(p23), +9, del(12)(p12), −14, −16, −18, −21, +mar1,+mar2[cp44]

[0089] Progression of LAPC-4 Xenograft to Androgen Independence

[0090] Prostate cancer cells are exquisitely sensitive to the growthstimulatory effects of androgen, but androgen-independent diseaseeventually develops in patients under the selective pressure of androgendeprivation. The mechanism for this transition to androgen-independentgrowth is unknown. The question of whether this phase of the diseasecould be modeled in SCID mice was determined using the LAPC-4 xenograft.which reproducibly forms tumors on serial passages in male mice with100% frequency. The androgen dependence of the xenograft was measured invivo by comparing the growth rates after implantation in intact malemice with those from castrated male mice or female mice. For LAPC-4, theaverage time for tumor formation in castrated male mice or female mice(n=10) was 13.4 weeks versus 4.3 weeks in intact males (n=14) (FIG. 3).The delayed growth in female mice was reversed by implantation of a90-day sustained-release testosterone pellet (FIG. 3). Theandrogen-independence of tumors growing in female or castrated male micewas confirmed by secondary transfer experiments. Once established, thesetumors grew within 4-5 weeks in.male, female and castrated male mice.

[0091] The LAPC-3 xenograft showed growth characteristics similar to theandrogen independent sublines of LAPC-4. After an initial latent periodfor passage 1, LAPC-3 tumors grew within 7-8 weeks regardless of thehormonal background of the recipient (FIG. 3), clearly establishing thisxenograft as androgen independent.

[0092] Clinically, anti-androgen therapy causes temporary regression ofdisease in most patients with advanced prostate cancer. To determine ifa similar phenomenon is observed in the mouse model, the effect of acuteandrogen deprivation on established tumors growing in male mice wasexamined. Equivalent size implants of the LAPC-4 xenograft were passagedinto a cohort of 14 male mice, all of which developed easily measurabletumors after four weeks. Half of these mice underwent castration, thenthe tumor sizes in each group were determined weekly by calipermeasurement of tumor diameters in three dimensions. Tumors in theuncastrated mice doubled in size over a 2-3 week period (FIG. 4). Incontrast, the castrated mice showed a decrease in tumor size at one weekof approximately 50 percent which persisted for 2-3 weeks These tumorsresumed growth after a variable latent period (3-8 weeks) and eventuallygrew to the same size seen in uncastrated mice. These results show thatthe LAPC-4 xenograft displays androgen-dependent growth, thatandrogen-independent sublines can be developed, and that this xenograftsimulates the clinical transition from androgen-sensitive toandrogen-independent disease.

[0093] LAPC-3 and LAPC-4 Express Wild-Type Androgen Receptors

[0094] To determine whether similar mutations are present in LAPC-3 andLAPC-4, exons 2-8 of the androgen receptor gene, which span the DNAbinding and ligand binding domains of the receptor, were sequenced.Single-strand conformational polymorphism (SSCP) analysis was alsoperformed. Each exon was amplified by PCR from genomic DNA of early andlate passage tumors and analyzed using previously characterized mutantand wild-type androgen receptor DNA as positive controls (Sutherland etal., 1996) The results show that both LAPC-3 and LAPC-4 containwild-type sequences in exons 2-8 Furthermore, these sequences remainwild-type in androgen independent LAPC-4 sublines. Immunoblot analysisconfirmed expression of androgen receptor protein of the appropriatesize. These results provide definitive evidence that androgenindependent prostate cancer progression can occur in the absence ofandrogen receptor mutations in the DNA or ligand binding domains.

[0095] LAPC-4 Cells Can Be Efficiently Transduced With Retroviruses:LAPC-4 xenograft cells can be successfully transduced by retrovirusespackaged transiently in 293T cells with an amphotropic envelope protein.LAPC-4 cells were infected with retrovirus stocks expressing the cellsurface Thy-1 protein and expression detected by flow cytometry using anantibody to Thy-1. The results showed Thy-1 expression in up to 50% ofthe cells 48 hours after infection, indicating successfulretroviral-mediated transduction of the Thy-1 gene into LAPC-4 cells.

Examples 2 Preparation of Single Cell Suspensions of Xenograft Cells

[0096] Materials and Methods

[0097] Single cell suspensions of subcutaneous LAPC-4 tumors wereprepared as follows. After removing xenograft tissue from SCID mouse,tissue was minced into 1-2 mm³ sections while the tissue was bathed in1× Iscoves medium, minced tissue was then centrifuged at 1.3K rpm for 4minutes, the supernatant was resuspended in 10 ml ice cold 1× Iscovesmedium and centrifuged at 1.3K rpm for 4 minutes. The pellet was thenresuspended in 1× Iscoves with 0.1% pronase E and incubated for 18minutes at room temperature with mild rocking agitation followed byincubation on ice for 2-4 minutes. The mixture was then filtered using a200 μm nylon mesh filer. Filtrate was centrifuged at 1.3K rmp for 4minutes, and the pronase was removed from the aspirated pellet byresuspending in 10 ml Iscoves and re-centrifuging. Resulting pelletswere resuspended in PrEGM pre-incubated at 37 degrees C. Cell countswere determined, and limiting dilutions were formulated as indicated inFIG. 5.

[0098] Results

[0099] The results of a limiting dilution analysis of tumor engraftmentusing single cell suspension of LAPC-4 xenograft cells are shown in(FIG. 5). The results show that single cell suspensions of xenograftcells can form subcutaneous tumors in male mice after injection of asfew as 10 LAPC-4 cells and that these cells retain the androgenresponsiveness of the parental tumors.

Example 3 Simulation Progression To Micrometastasis in SCID Mice BearingSubcutaneous Tumors

[0100] Materials and Methods

[0101] The LAPC-4 xenograft was used in this study. This xenograft wasderived from a lymph node containing metastatic prostate cancer cells,and 100% of male mice inoculated subcutaneously with LAPC-4 cellsdevelop localized tumors after 4-6 weeks without evidence of bonymetastasis. The presence of micrometastasis in SCID mice implanted withLAPC-4 tumors was determined by analyzing the peripheral blood forprostate cancer cells using RT-PCR assays for PSA mRNA. SimultaneousRNA-PCR studies using β-actin primers demonstrated equivalent RNAloading. To confirm that positive PSA mRNA signals were not due tocontamination with tumor cells during the necropsy procedure of duringthe preparation of RNA, samples were simultaneously prepared from acontrol mouse that was not implanted with a xenograft. No PSA expressionwas detected in control mice, even after prolonged autoradiographexposure times (FIG. 6).

[0102] Bone marrow, spleen, liver, lung and kidney tissue from miceimplanted with subcutaneous LAPC-4 tumors was also analyzed for thepresence of prostate cancer cells using RT-PCR to detect PSA mRNA.

[0103] Results

[0104] Examples of the analysis from two mice (FIG. 6A, mouse nos. 213and 241) demonstrate detection of PSA mRNA in blood at a 0.1-1.0% level,which is comparable to levels reported in clinical studies. Other organswere positive in several mice, including bone marrow (mouse 213, 241),lung (mouse 214), and spleen (data not shown). The results from 12animals bearing LAPC-4 xenografts (Table 4) show that 50 percent of micehave PSA mRNA positive cells (level of PSA expression by RT-PCR of 0.1percent or greater) detected in peripheral blood, bone marrow or spleen.The level of expression was roughly quantitated by comparison to aseries of LNCaP cells diluted into murine fibroblasts and varied from0.1% to 1.0%. It is of interest that the frequency of detectingmicrometastatic disease was higher (80%) in female mice or in male micecastrated prior to implantation compared to intact males (27%). Theseresults suggest that the transition to androgen-independent disease isassociated with a higher metastatic rate, a hypothesis which is alsosupported by clinical experience. TABLE 4 FREQUENCY OF DETECTION OF PSAPOSITIVE CELLS IN HEMATOPOETIC TISSUES OF LAPC-4 BEARING SCID MICENumber of mice with PSA positive cells in hematopoietic Group organs pertotal number analyzed Intact Males  2/7 (29%) Castrate Males  4/5 (80%)(or Females) Total 6/12 (50%)

Example 4 Generation of Intraprostatic Tumors With Xenograft Cells

[0105] Materials and Methods

[0106] Single cell suspensions were prepared from subcutaneousxenografts as described in Example 2. SCID mice were anesthetized withKetamine/Xylazine prior to implantation. Transverse incisions were madein the lower abdomen of mice, abdominal wall muscles were incised, andthe bladder and seminal vesicles were delivered through the incision toexpose the dorsal prostate. Approximately 10,000 LAPC-4 suspended in 10μl PrEGM were slowly injected into the dorsal prostate under the capsulevia a 30 gauge needle, and the incisions closed using a running suture.

[0107] Results

[0108] Intraprostatic injection of single cell suspensions prepared fromthe LAPC-4 and LAPC-9 xenografts and from the LAPC-4 cell line resultedin orthotopic tumors in recipient SCID mice with 100% efficiency.

Examples 5 Simulation of Progression to Metastatic Stage of ProstateCancer in SCID Mice Bearing Intraprostatic Tumors

[0109] Materials and Methods

[0110] Single cell suspensions of LAPC-4 xenograft cells were preparedand used to establish orthotopic tumors in the prostates of SCID mice asdescribed in the preceding example. The presence of metastases weredetermined by histologic examination and by RT-PCR to detect PSA mRNAbetween 8 and 12 weeks post-injection.

[0111] Results

[0112] The results, shown in Table 5 below, indicate high frequencies oflymph and pulmonary metastasis as well as a significant frequency ofbone marrow metastasis formation. An enhanced frequency of bonemetastasis was observed in a subset of the mice pretreated with acombination of radiation and NK cell depletion. Similar results wereobtained using the LAPC-9 xenograft. TABLE 5 PATTERN OF METASTASIS AFTERORTHOTOPIC INJECTION OF LAPC-4 TUMOR SITE FREQUENCY Local Tumor 100% Pelvic Lymph Nodes 90% Lung 90% Bone Marrow 30%

Example 6 Simulation of Progression to Osteoblastic Bone Metastasis inSCID Mice Inoculated Intratibially With Single Cell Suspension ofXenograft Cells

[0113] Materials and Methods

[0114] Tibial Injection Assay: Prostate cancer cells were isolated froma subcutaneous xenograft LAPC-4 tumor and prepared as a single cellsuspensions as described in Example 2. Ten thousand LAPC-4 cellssuspended in 1 μl Matrigel were surgically injected into each proximaltibial metaphyses of a cohort of SCID mice via a 27 gauge needle. Threemice were sacrificed at each of 2, 4, 6, 8 and 12 weeks post injection.Serum PSA levels were periodically assayed by ELISA. At 2 weeks, frozenbone sections were analyzed immunohistochemically for cytokeratin-18staining with an antibody specific for human cytokeratin-18 or anisotype control antibody. Longitudinal sections of tibias from micesacrificed at 4, 6 and 8 weeks were analyzed for tumor growth byhematoxylin and eosin (H+E) staining of decalcified paraffin sections.Radiographs of mice were taken at necropsy to monitor evidence ofosteoblastic bone lesions.

[0115] Results

[0116] At 2 weeks, small numbers of human prostate cancer cells werevisualized by immunohistochemical staining with anti-cytokeratin-18antibody (FIG. 7). Cytokeratin-18 positive cells were observed scatteredthroughout the medulary cavity. This data indicates that the majority ofLAPC-4 cells injected into the mouse tibia either die or migrate toother locations since only a small subset of the injected cells can bedetected at this time point.

[0117] At 4 weeks, small foci of tumor growth were observed in a fewisolated areas, usually adjacent to normal bone spicules, by H+Ehistology (FIG. 8A) and PSA could be detected in serum. At the 6 and 8week time points, more extensive tumor growth throughout the marrowcavity was observed together with a progressive increase in new boneformation indicative of osteoblastic activity within the marrow cavityin response to surrounding tumor cells (FIG. 8B and C). Serum PSA levelswere markedly elevated at this time point.

[0118] By 8 weeks, bone lesions were visible radiographically as amixture of osteoblastic and osteolytic lesions with dominant boneformation similar to clinical observations in human prostate cancer.Referring to FIG. 9, the left panel shows a radiograph of a normal mousetibia with sharp, well defined cortex and relatively radioopaque marrowcavity. The right panel is a radiograph of the marrow cavity of thetibia injected with LAPC-4 xenograft cells, showing a heterogeneousincrease in bone density due to osteoblastic activity and destruction ofone area of the cortex. These results indicate that LAPC-4 xenograftcells can proliferate in murine bones, suggesting that the crosstalkbetween bone stroma and prostate cancer cells can occur across species.

Example 7 Isolation of Prostate Cancer Cells From Bone Marrow of SCIDMice Bearing Subcutaneous Xenografts

[0119] The presence the cell surface protein galectin-6 on LAPC-4 cellswas established by incubating intact LAPC-4 cells with a human specificmonoclonal antibody to galectin 6 or isotype control. The antibody wasvisualized by flow cytometry following incubation with a secondaryantibody conjugated to FITC. The flow cytometry results show expressionof galectin-6 at a level that is at least one order of magnitude abovebackground (FIG. 10). Similar experiments performed on mouse bone marrowshowed no galectin staining.

[0120] As described in Example 3, small numbers of prostate cancer cellscan be detected in the bone marrow of SCID mice bearing subcutaneousxenografts at 4-6 weeks post-inoculation, representing somewhat lessthan 1% of the cells in the marrow. This population of prostate cancercells may be isolated from bone marrow, using Miltenyi Magnetic Minimacs(Sunnyvale, Calif.) antibody-based affinity purification system andanti-galectin-6 antibody as follows. Twenty mice bearing subcutaneousLAPC-4 tumors are euthanized 4-6 weeks after implantation of xenografts.Bone marrow is harvested from the tibias and femurs by flushing marrowcavities with saline. Marrow is pooled and incubated with a humanspecific monoclonal antibody to galectin-6 and a secondary antibodyconjugated to magnetic beads and run through the Minimacs column asrecommended by the manufacturer. LAPC-4 cells will be retained on thecolumn, while mouse bone marrow cells will pass through. Purified LAPC-4cells may then be harvested from the column and expanded by seedingsubcutaneous tumors in SCID mice.

Example 8 Isolation of Prostate Cancer Cells From Bone Marrow of SCIDMice Injected Intratibially With Xenograft Cells

[0121] Intratibial tumors are established in SCID mice using LAPC-4cells as described in Example 6. LAPC-4 cells growing in bone marrow arerecovered from mice after necropsy at 12 weeks by flushing the tibialmarrow cavity with saline and harvesting the cells. At 12 weekspost-injection, about 90% of the recovered cells are prostate tumorcells with some residual murine bone marrow cells. This population ofcells may be further purified for prostate cancer cells using agalectin-6 antibody/magnetic affinity purification approach as describedin Example 7.

Example 9 LAPC-4 Cell Line Retains Expression of PSA, Androgen Receptor,and Prostatic Acid Phosphatase Through Multiple Passages

[0122] Materials And Methods

[0123] A continuous cell line was established from the LAPC-4 xenograftby serial passage of trypsinized, minced xenograft tissue in Iscove'sgrowth medium supplemented with 20% fetal calf serum.

[0124] Results

[0125] LAPC-4 cells growing in continuous culture in vitro have retainedexpression of PSA, androgen receptor, and prostatic acid phosphatasethrough more than 20 passages. In addition, LAPC-4 cells contain nomutations in either the DNA or ligand binding domains of the androgenreceptor, which is a novel characteristic among known prostate cancermodels. The only other PSA-expressing cell line, LNCaP, expresses anandrogen receptor with a point mutation in the ligand binding domain. Inaddition, LAPC-4 cells continue to express androgen receptor in androgenindependent sublines, analogous to results obtained from the analysis ofclinical material. The LAPC-4 cell line is androgen dependent sincetumors grow rapidly in male mice but not in female or castrated malemice. The LAPC-4 cell line has remained established for more than 20passages and has been in continuous culture for over 18 months. Thesecells continue to express PSA, form tumors in SCID mice, and retainandrogen-responsiveness.

Example 10 Testing The Biological Effects of Candidate Genes on AndrogenIndependent Growth in Vivo

[0126] Some genes upregulated in hormone refractory prostate cancer maycontribute to the pathogenesis of androgen independence. Bcl-2, forexample, which is upregulated in many advanced prostate cancers, hasbeen demonstrated to confer androgen independence to androgen dependentLNCaP prostate cancer cell line (Raffo et al., 1995); In accordance withthis example, one can access in vivo the contribution of candidate genesto the androgen independent phenotype.

[0127] LAPC-4 Androgen Dependent Tumor Explants Grow in Tissue Cultureand Form Androgen Dependent Tumors Upon Reinjection Into SCID Mice

[0128] Current bioassays for androgen dependent and independent growthrely almost exclusively on the LNCaP prostate cancer cell line, becauseit is the only cell line available which displays features of androgendependence. In order to circumvent the problem of long-term passagedcell line with the potential for multiple in vitro mutations, the LAPC-4xenograft was grown in short term culture and then reinjected into miceto form tumors. Explanted tumors were then manipulated genetically andthe effects of these manipulations were measured in vivo.

[0129] LAPC-4 tumors were minced into small pieces and cultured in mediawith 15% fetal calf serum. Outgrowth of both epithelial cells andfibroblasts was noted after 2-3 days. Cells then grew to confluence andcould be successfully passaged to remove the original tumor pieces.RT-PCR confirmed continued PSA expression. 1×10⁷ cells were thenreinjected into either intact male or castrated SCID mice. Similar tothe initial experiments, injected cells formed tumors in an androgendependent fashion, requiring prolonged periods to form tumors incastrated mice.

[0130] LAPC-4 Cultures Can Be Transduced With Retrovirus

[0131] In order to test the infectability of explanted LAPC-4 cells byretrovirus, these cells were transduced with a retroviral vectorcontaining a truncated nerve growth factor receptor gene (NGFR). A PG13packaging cell line, containing the gibbon-ape leukemia virus (GALV)envelope, was used to generate high titer virus. Retrovirus virionsproduced in this manner have the unique property of infecting human, butnot murine, cells, thus avoiding introduction of transgene into mousestromal cells (Bauer et al., 1995). After infection, the cells werestained with an antibody directed against NGFR and analyzed by FACSanalysis. Five-10% of cells were transduced. Murine fibroblasts negativecontrols showed no infection, while human 293T cells were efficientlytransduced.

[0132] Biological Assays For Cdnas Upregulated in Androgen-IndependentProstate Cancer

[0133] Candidate cDNAs can be cloned into the 5′ position of theretroviral vector pSRalpha used extensively in our laboratory (Afar etal., 1994). A reporter gene, either NGFR, LacZ, or human codon-optimizedgreen fluorescent protein (GFP), would be inserted downstream. Theplasmid can be transfected into the PG13 packaging cell line, viruscollected, and titers measured. LAPC-4 cells can be infected after thefirst passage and then expanded without selection until sufficientnumbers are available for injection. Transgene expression can beconfirmed either by FACS analysis or by northern blot analysis using theRDA cDNA clone as a probe.

[0134] Two different types of experiments can be performed. In thefirst, infected cells are injected in to the flanks of intact male SCIDmice. After tumors form in both flanks of an individual mouse, one tumoris removed and the mouse is then castrated. The explanted tumor isanalyzed to quantify the percentage of cells infected. This can be doneeither by LacZ straining or by FACS analysis for GFP or NGFR. Weanticipate that 5-10% of cells will carry the transgene. The remainingtumor can be similarly analyzed after it regresses and regrows (i.e.,about 4-8 weeks after castration). If the transgene confers a survivaladvantage or androgen independence to infected cells, we would expect tosee the percentage of cells carrying the transgene to increase afterhormone ablation Multiple mice can be injected with each construct andpositive results confirmed by repetition.

[0135] In a second set of experiments, one can implant infected cellsinto intact and castrated male mice in parallel after quantifyinginfection frequency. Resulting tumors (at 4 and 12 weeks, respectively)are analyzed for insert frequency as described above. Again. we expectthat “androgen independent” genes will provide an androgen independentgrowth advantage and predominate in the resulting tumor. In addition, itis possible thy a given candidate gene will shorten the time to tumorformation in castrated males. This can also be measured. Finally, it ispossible that a given gene could cause aggressive androgen dependentgrowth. This too can be quantified in this assay, by comparing time totumor formation and insert frequency before and after injection intointact male mice.

[0136] These assays can be validated with positive controls. Inparticular, one can use bcl-2, c-myc, and c-met, since these have beenconsistently associated with androger independence.

[0137] The present invention is not to be limited in scope by theembodiments disclosed herein, which are intended as single illustrationsof individual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

[0138] References

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What is claimed is:
 1. An immune deficient mouse having a human prostatecancer xenograft of locally advanced or metastatic prostate cancer. 2.The mouse of claim 1, wherein the locally advanced prostate cancer is atstage C.
 3. The mouse of claim 1, wherein the metastatic prostate canceris at stage D.
 4. A SCID mouse of claim
 1. 5. The mouse of claim 1,wherein the xenograft is androgen dependent.
 6. The mouse of claim 1,wherein the xenograft is androgen independent.
 7. The mouse of claim 1,wherein the xenograft is androgen dependent in the presence of androgenand is androgen independent in the absence of androgen.
 8. The mouse ofclaim 1, wherein the xenograft is derived from an explant selected fromprostate, lymph node, lung or bone tissue.
 9. A method of generating ahuman prostate cancer xenograft that simulates prostate cancer in micecomprising implanting locally advanced or metastatic prostate cancertissue or cell suspension thereof from a human in an immune deficientmouse and allowing the tissue so implanted to grow.
 10. The method ofclaim 9, wherein the tissue is implanted subcutaneously.
 11. The methodof claim 9, wherein the xenograft so grown is implanted into a secondmouse and allowing the xenograft to grow.
 12. A method of simulating theprogression of human prostate cancer from primary tumor formation tomicrometastasis in an animal model comprising: a. generating a humanprostate cancer xenograft in an immune deficient mouse by the method ofclaim 9; and b. allowing the xenograft tumor to grow for a timesufficient to permit the detection of prostate cancer cells not withinthe implant site in the immune deficient mouse.
 13. The method of claim12, wherein the xenograft in (a) is implanted subcutaneously.
 14. Themethod of claim 12, wherein the xenograft in (a) is implantedintraprostatically.
 15. The method of claim 12, wherein detection iseffected in the peripheral blood of the immune deficient mouse.
 16. Themethod of claim 12, wherein detection is effected in the bone marrow ofthe immune deficient mouse.
 17. A method of simulating the progressionof osteoblastic bone metastasis in human prostate cancer comprising: a.injecting a single cell suspension of prostate cancer cells preparedfrom a prostate cancer xenograft generated by the method of claim 9 intothe tibial bone marrow cavity of an immune deficient mouse; and b.allowing the injected cells to grow and form an osteoblastic bone lesionwhich simulates the progression of osteoblastic bone metastasis in humanprostate cancer.
 18. A SCID mouse produced by the method of claim
 9. 19.An assay for assessing the effect of a treatment for human prostatecancer comprising: (a) applying the treatment to an immune deficientmouse bearing a subcutaneous human prostate cancer xenograft generatedby the method of claim 9; and, (b) determining the effect of thetreatment on the growth of the xenograft.
 20. An assay for determiningthe effect of a gene on the progression of micrometastatic prostatecancer comprising: (a) generating a subcutaneous prostate cancerxenograft in an immune deficient mouse by the method of claim 9; (b)transducing the cells of the xenograft with the gene in vivo; (c)evaluating the presence of micrometastasis in the immune deficient mouseby detecting prostate cancer cells in the peripheral blood, bone marrow,lymph nodes or other sites distant from the site of the subcutaneousxenograft; wherein the effect of the gene on the progression ofmicrometastatic prostate cancer is determined by reference to a controlimmune deficient mouse bearing a subcutaneous human prostate xenograftgenerated with a untransduced subset of the isolated cells.